Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen commonly associated with traveler's diarrhea, infecting approximately 280 million people annually. Although most cases are not life threatening, it is estimated that 300,000 to 700,000 infants and children are killed by this pathogen each year. Dehydrating diarrhea is produced when the pathogen elaborates the heat-labile (LT) and/or the heat-stable (ST) toxin. In the decades since their discovery both enterotoxins have been extensively characterized. The other well characterized category of ETEC virulence factors are the adhesins; both fimbrial and afimbrial. Although there are exceptions; the vast majority of research in this field involves some aspect of LT, ST, or adhesin biology/epidemiology. However we have recently discovered a new ETEC virulence factor. We propose that this virulence factor is a novel third enterotoxin that enhances ETEC virulence by blocking the activation of a newly described effector of innate immunity [Perforin-2 (P2)]. This enteroxotin is deployed by an atypical Type 1 Secretion System that accepts its substrate from the periplasm rather than the cytosol. In Aim 1 we will characterize the virulence factor's mechanism of action and cellular target(s). In Aim 2 we will characterize its secretory pathway. By elucidating the molecular mechanism(s) of this novel virulence factor we will gain new insight into ETEC pathogenesis which may lead to novel therapeutics to reduce the global burden of ETEC infections. These studies will also provide new knowledge of an atypical protein secretion system that may be applicable to other bacterial pathogens. In addition these studies will enhance our understanding of P2; an important but under characterized component of the innate immune system.